Physics (Internal)

Embryoid Body Shell Formation Reduces Diffusive Transport: new strategies fro stem cell differentiation

This event is part of the Biophysics/Condensed Matter Seminar Series.

Abstract: Human embryonic stem (hES) cells have the ability to differentiate into all types of cells. Differentiation of hES cells into cells with therapeutic potential is often initiated by embryoid body (EB ) formation. Embryoid bodies begin as three-dimensional hES cell aggregates that differentiate, recapitulating the early stages of embryonic development. Differentiation of hES cells towards specific lineages is often assisted by treating EBs with soluble biochemicals such as cytokines, growth factors and vitamins. However, treatment with exogenous biochemicals has shown limited potential. As a consequence, differentiation strategies have limited dosing regimens. We have conducted a scanning electron microscopy analysis over 14 days. We revealed the time-dependent changes in EB structure which led to the formation of a shell that significantly reduces the diffusive transport of a model molecule (374 Da) by >80%. We found that the shell consists of a cellular network interwoven with extracellular matrix material, of which collagen is identified as a component. Disrupting the collagen-rich shell restored diffusive uptake to more than 40%. We tested the impact of increased diffusive uptake in EBs using retinoic acid as the soluble biochemical for inducing neuronal differentiation. Flow cytometry and quantitative RT-PCR analysis of EBs treated with a 15 minute collagenase digestion exhibited an increase in gene expression of neural cell adhesion molecule (NCAM) relative to untreated EBs. This indicates that increased diffusive transport due to collagenase permeabilization promotes the differentiation of neuronal cells. Our results suggest that limitations in the diffusive transport of biochemicals need to be considered when formulating EB differentiation strategies.